The advent of recombinant DNA technology has opened the door for many protein-based biological therapies. In most circumstances, these protein therapeutics are produced by cells, highly purified, and prepared for administration to patients. Often, the recombinant DNA encoding the therapeutic protein, must be transfected into the protein-producing cells. Viruses can remain in the culture after transfection and contaminate the protein samples. Additionally, cells used for expressing proteins of interest may encode viral genomes in their DNA or otherwise contain endogenous viruses, which is another potential source of contamination to a therapeutic product derived from cells. Therefore, biologically-derived therapeutics must undergo at least two robust virus purification steps in order to meet the safety requirements of regulatory agencies such as the FDA to ensure no active viruses are administered to a patient.
There are several methods known in the art to inactivate viruses. Treatment with low pH, the use of detergents, salts, and heat inactivation have all been used to inactivate viruses in protein preparations, but each method has its own disadvantages, and may not be suitable or optimal for some protein products as discussed in further detail below.
Low pH has been used to inactivate viruses as in U.S. Pat. No. 6,955,917, but this has the potential to precipitate proteins, cause aggregation of the product, and/or alter the conformation of certain proteins which can lead to product loss.
EP0131740 B1 describes a method for the inactivation of lipid-coated viruses in compositions containing labile proteins. The method described in EP 0131740 B1 consists of contacting the composition containing labile protein with an effective amount of a dialkyl or trialkyl phosphate for a period of time sufficient to render the composition containing labile protein free of lipid-containing viruses.
U.S. Pat. No. 6,528,246 B2 describes a method for inactivation of viruses using combinations of tri-n-butyl phosphate and Tween, or sodium cholate/TNBP (tri-n-butyl phosphate) and other buffers, detergents and/or surfactants, but requires the use of high concentrations of auxiliary agents such as saccharose and also heat inactivation in the range of 55° C. to 67° C. which can denature certain proteins and lead to degradation resulting in loss of product.
Other detergents such as TRITON® X-100 (Sigma-Aldrich Corp., St. Louis, Mo., USA) have been used to inactivate viruses, but present problems with high amounts of waste product when used on an industrial scale. In the examples of WO 94/26287, a “detergent/salting-out” method is applied to three isolated proteins in solution, which are transferrin, antithrombin III and albumin. If the TRITON® X-100 method is applied under conditions such that the yield of the target protein is not substantially affected, frequently the concentration of TRITON® in the product is still very high. In example 4 of WO 94/26287, the inventors recovered 95% of albumin, but obtained a product comprising 250 ppm TRITON® X-100 and 35 ppm TNBP. Especially when producing medical preparations, TRITON® X-100 concentrations above 50 ppm, or even above 10 ppm are preferably avoided, and it is generally desirable to reduce the detergent content as much as possible. Additionally, some therapeutic proteins are inactivated by TRITON® X-100 and, thus, this method for virus inactivation is not optimal for many protein products.
Accordingly, there is a need in the art to inexpensively and safely inactivate or reduce infectious virus titers while preserving the integrity, biological, and/or therapeutic activity of the protein product.
Arginine is unique among naturally occurring amino acids in that it has been found to prevent protein aggregation and suppress protein interactions without substantially altering protein conformation. Given these attributes, 0.1M to 1M arginine has been used to facilitate refolding of recombinant proteins solubilized from inclusion bodies and 0.5 to 2M arginine has been used to extract active, folded proteins from insoluble pelleted material expressed as a recombinant product in E. coli. (Tsumoto et al., Biotechnology., 20, 1301-1308 (2004); Ishibashi et al., Protein Expression and Purification, 42, 1-6 (2005); Arakawa et al., Biophysical Chemistry, 127, 1-8 (2008)). Arginine has also been used to enhance recovery of proteins from various types of chromatographic media such as in Protein-A, gel permeation, and dye-affinity chromatography. (Arakawa et al., Protein Expression and Purification, 54, 110-116 (2007); Ejima et al., Analytical Biochemistry, 345, 250-257 (2005)). Arginine has also been used as one component in protein stabilizing formulations, for example, to protect proteins from being inactivated during heat treatment procedures. (Miyano, et al., U.S. Pat. No. 5,116,950, issued May 26, 1992).
Kozloff et al. have observed that use of 0.2M arginine irreversibly inactivated preparations of some T-even strains of bacteriophage (T2L, a non-enveloped virus). Kozloff et al. also found that this virus specific inactivation was most effective at 30° C. and in a pH range of 6.5 to 8.25, and could be accomplished with arginine at 0.033 to 0.2M. However, arginine inactivation of T2L was increasingly ineffective at concentrations of above 0.4 M. Kozloff et al. also observed that arginine did not inactivate T-odd strains of bacteriophage. This difference is presumably due to differences in tail structures of T-even versus T-odd bacteriophage (with which arginine apparently specifically interacts to bring about T-even inactivation). Kozloff et al., Jour. Virol., 3(2), 217-227 (1969).
Yamasaki et al. have observed that at a low (acidic) pH and at low temperatures (samples on ice), arginine can inactivate the enveloped herpes simplex virus type-1 (HSV-1) and influenza virus. However, at more neutral pH levels (i.e., pH 5.0-pH 7.0), Yamasaki et al. found arginine to be ineffective at inactivating these viruses. Yamasaki et al. also found that arginine was ineffective at inactivating non-enveloped polio virus. (Yamaski et al., Journal of Pharmaceutical Sciences, (Jan. 10, 2008) 97(8), 3067-3073).